Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda.

BACKGROUND The distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use. METHODS The WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated. RESULTS The WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage. CONCLUSIONS The WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation.

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.

La dénervation rénale unilatérale et le système kallikréine-bradykinine rénal chez le rat

But de l’étude L’effet antihypertenseur de la dénervation rénale chez les patients hypertendus s’explique partiellement par une augmentation de la natriurèse tubulaire. Pour étudier une contribution possible du système kallikréine-kinines (SKK) à cette natriurèse dans le rat, nous avons dosé dans le plasma et dans les tissus l’activité de la kallikréine (AK) et la concentration de la bradykinine (BK). Méthodes Pour AK, nous avons adapté et validé un essai enzymatique qui libère la para-nitroaniline à partir du tripeptide H-D-Pro-Phe-Arg-pNA ; les coefficients de variation (CV) intra-essai et inter-essai étaient inférieurs à 8 % pour AK plasmatique et tissulaire (plasma n = 6 et 13, tissu n = 4). La linéarité d’une série de dilutions confirmait la spécificité de l’essai. Le dosage de BK tissulaire se basait sur une méthode établie pour le plasma : tissus étaient homogénéisés et BK extraite et isolée par éthanol et HPLC, et finalement quantifiée par radio-immunoessai. Les CV intra- et inter-essai pour BK étaient 18 % dans le plasma (n = 8 et n = 35) et inférieurs à 16 % dans différents tissus (n = 5–8). Résultats Chez le rat mâle Wistar (n = 3), la BK plasmatique était de 8,2 ± 6,6 fmol/mL (M ± SD) et la BK tissulaire (fmol/g) variait, pour les 14 organes testés, de 14 ± 3 pour le cerveau à 521 ± 315 pour la glande sous-maxillaire. Six jours après dénervation rénale gauche, la BK rénale gauche (89 ± 9) n’était pas différente comparée à la BK rénale droite (75 ± 23). De même, l’AK était identique dans les deux reins (gauche 18,0 ± 1,5, droit 15,8 ± 1,4 μkat/g). Conclusion Un effet éventuel de la dénervation rénale unilatéral sur le SKK rénal devrait donc être bilatéral.

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex

The European trout (Salmo trutta species complex) is genetically very diverse consisting of five distinct mitochondrial lineages that probably originated in the Pleistocene. Here, we describe a novel pyrosequencing protocol to generate two short sequence reads from the mitochondrial control region, which allow the unambiguous identification of all five lineages. The approach was found to be easily transferable between laboratories and should be a valuable tool for the assessment of genetic diversity in trout. Pyrosequencing-based assays for molecular species identification are expected to be generally useful whenever multiple positions in a short DNA sequence need to be assessed.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests A study in 9 Swiss laboratories

INTRODUCTION: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43?g/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).

Improved sensitivity of an interferon-gamma release assay (T-SPOT.TB™) in combination with tuberculin skin test for the diagnosis of latent tuberculosis in the presence of HIV co-infection

Background Interferon-gamma release assays (IGRA) are more specific than the tuberculin skin test (TST) for the diagnosis of Mycobacterium tuberculosis infection. Data on sensitivity are controversial in HIV infection. Methods IGRA (T-SPOT.TB) was performed using lymphocytes stored within 6 months before culture-confirmed tuberculosis was diagnosed in HIV-infected individuals in the Swiss HIV Cohort Study. Results 64 individuals (69% males, 45% of non-white ethnicity, median age 35 years (interquartile range [IQR] 31-42), 28% with prior AIDS) were analysed. Median CD4 cell count was 223 cells/μl (IQR 103-339), HIV-RNA was 4.7 log10 copies/mL (IQR 4.3-5.2). T-SPOT.TB resulted positive in 25 patients (39%), negative in 18 (28%) and indeterminate in 21 (33%), corresponding to a sensitivity of 39% (95% CI 27-51%) if all test results were considered, and 58% (95% CI 43-74%) if indeterminate results were excluded. Sensitivity of IGRA was independent of CD4 cell count (p = 0.698). Among 44 individuals with available TST, 22 (50%) had a positive TST. Agreement between TST and IGRA was 57% (kappa = 0.14, p = 0.177), and in 34% (10/29) both tests were positive. Combining TST and IGRA (at least one test positive) resulted in an improved sensitivity of 67% (95% CI 52-81%). In multivariate analysis, older age was associated with negative results of TST and T-SPOT.TB (OR 3.07, 95% CI 1,22-7.74, p = 0.017, per 10 years older). Conclusions T-SPOT.TB and TST have similar sensitivity to detect latent TB in HIV-infected individuals. Combining TST and IGRA may help clinicians to better select HIV-infected individuals with latent tuberculosis who qualify for preventive treatment.

Twelve years' detection of respiratory viruses by immunofluorescence in hospitalised children: impact of the introduction of a new respiratory picornavirus assay

Background Direct immunofluorescence assays (DFA) are a rapid and inexpensive method for the detection of respiratory viruses and may therefore be used for surveillance. Few epidemiological studies have been published based solely on DFA and none included respiratory picornaviruses and human metapneumovirus (hMPV). We wished to evaluate the use of DFA for epidemiological studies with a long-term observation of respiratory viruses that includes both respiratory picornaviruses and hMPV. Methods Since 1998 all children hospitalized with respiratory illness at the University Hospital Bern have been screened with DFA for common respiratory viruses including adenovirus, respiratory syncytial virus (RSV), influenza A and B, and parainfluenza virus 1-3. In 2006 assays for respiratory picornaviruses and hMPV were added. Here we describe the epidemiological pattern for these respiratory viruses detected by DFA in 10'629 nasopharyngeal aspirates collected from 8'285 patients during a 12-year period (1998-2010). Results Addition of assays for respiratory picornaviruses and hMPV raised the proportion of positive DFA results from 35% to 58% (p < 0.0001). Respiratory picornaviruses were the most common viruses detected among patients ≥1 year old. The seasonal patterns and age distribution for the studied viruses agreed well with those reported in the literature. In 2010, an hMPV epidemic of unexpected size was observed. Conclusions DFA is a valid, rapid, flexible and inexpensive method. The addition of assays for respiratory picornaviruses and hMPV broadens its range of viral detection. DFA is, even in the "PCR era", a particularly adapted method for the long term surveillance of respiratory viruses in a pediatric population.